IgE plays a central role in allergic disorders. IgE high affinity receptors (FcεRI) are located on mast cells and basophils, which serve as antigenic targets stimulating the further release of inflammatory mediators producing many of the manifestations of allergic disease.
IgE-mediated inflammation occurs when antigen binds to the IgE antibodies that occupy the FcεRI receptor on mast cells. Within minutes, this binding causes the mast cell to degranulate releasing certain preformed mediators. Subsequently, the degranulated cell begins to synthesize and release additional mediators de novo. The result is a two-phase response: an initial immediate effect on blood vessels, smooth muscle, and glandular secretion (immediate hypersensitivity), followed up a few hours later by cellular infiltration of the involved site.
IgE antibodies bind to mast cells via the numerous high-affinity Fc receptors on the surface of each cell. The binding is noncovalent and reversible, so that bound antibodies are in constant equilibrium with the pool of circulating IgE. As a result, each mast cell can bind many different antigens. A response is initiated when a multivalent antigen binds two or more IgE antibodies occupying FcεRI receptors. The cross-linking transmits a signal that activates the mast cell, resulting in activation of protein tyrosine kinases and increasing the intracellular free calcium levels. Soon after, cytoplasmic granules fuse with one another and with the surface membrane, discharging their contents to the exterior. Basophils also express FcεRI receptors, but their effect on immediate hypersensitivity reactions is unknown.
This immediate phase of the inflammatory response results mainly from preformed mediators (especially histamine) that are stored in mast cell granules, as well as certain rapidly synthesized arachidonate derivatives. Maximal intensity of the response results after about 15 minutes after the initial antigen contact. Characteristics of this phase are erythema, localized edema in the form of a wheal and pruritus (itching). However, the granule contents also induce local expression of the vascular addressin VCAM-1 (Vascular Cell Adhesion Molecule) which assists in vascular permeability. Additional mast cell granule contents include RANTES and other chemokines, which are molecules having chemoattractant activity for leukocytes and fibroblasts, ultimately resulting in inflammation of the site.
Manifestations of the late phase are due in part to presynthesized TNF-α and in part to other mediators (e.g., PAF, IL-4 and arachidonate metabolites), the synthesize of which begins after mast cell degranulation. The effects of these mediators becomes apparent about six hours after antigen contact and are marked by an infiltrate of eosinophils and neutrophils. Clinical features of the late phase include erythema, induration, warmth, pruritus, and a burning sensation at the affected site. Mast cell-derived IL-4 promotes the production of TH2 cells. TNF-α not only functions in the short term as a leukocyte chemoattractant, but can also stimulate local angiogenesis, fibroblast proliferation, and scar formation during prolonged hypersensitivity reactions.
IgE-mediated inflammation is the mechanism underlying atopic allergy (such as hay fever, asthma and atopic dermatitis), systemic anaphylactic reactions and allergic urticaria (hives). It may normally play a role as a first line of immunologic defense, since it causes rapid vasodilation, facilitating entry of circulating soluble factors and cells to the site of antigen contact. Many of the most destructive attributes of allergic disease are due to the actions of the chemoattracted leukocytes, rather than from the mast cells themselves.
The use of bacteriophage particles for display of diverse peptide libraries (phage display) has been described (Lowman, (1997) Ann. Rev. Biophys. Biomol. Struct. 26:401–424). Phage display technology provides a means for generating both structurally constrained and unconstrained peptide libraries (Devin et al., (1990) Science 249:404–406; Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA 87:6378–6382; Lowman and Wells (1991) Methods: Comp. to Methods Enzymol. 3:205–216; Lowman (1997) Ann. Rev. Biophys. Biomol. Struct. 26:401–424). These libraries can be used in the identification and selection of peptide ligands that bind a predetermined target molecule (Lowman (1997), supra); Clackson and Wells (1994) Trends Biotechnol. 12:173–184; Devlin et al., (1990) supra). Phage display has been used to identify peptide motifs that home to a cellular target (Arap et al., (1998) Science 279:377–380); bind the human type I interleukin 1 (IL-1) receptor and block the binding of IL-1 (Yanofsky et al., (1996) Proc. Natl. Acad. Sci. USA, 93:7381–7386); bind to and activate the receptor for the cytokine erythropoietin (EPO)(Wrighton et al., (1996) Science 273:458–463); bind the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO)(Cwirla et al., (1997) Science, 276:1696–1699), bind and inhibit Factor Vila (Dennis et al. (2000) Nature 404:465) or to generate affinity improved or matured peptide ligands from native protein binding ligands (Lowman et al., (1991) Biochemistry 30: 10832–10838).
Using structurally constrained peptide libraries generated by monovalent phage display, 14 amino acid peptides that bind to insulin-like growth factor 1 binding proteins (IGFBPs) have been isolated (Lowman et al., (1998) Biochemistry, 37:8870–8878). The peptides contain a helix structure and bind IGFBPs in vitro liberating insulin like growth factor-a (IGF-1) activity (Lowman et al., (1998) supra). Utilizing in vivo phage selection, the technique has been used to identify and isolate peptides capable of mediating selective localization to various organs such as brain and kidney (Pasqualini and Ruoslohti (1996) Nature 380:364–366) as well as to identify peptides that home to particular tumor types bearing αvβ3 or αvβ5 integrins (Arap et al., (1998) Science 279:377–380). U.S. Pat. No. 5,627,263 describes peptides that are recognized by and selectively bind the α5β1 integrin. Examples of affinity or specificity improved proteins include human growth hormone, zinc fingers, protease inhibitors, ANP, and antibodies (Wells, J. and Lowman H. (1992) Curr. Opin. Struct. Biol. 2:597–604; Clackson, T. and Wells, J. (1994) Trends Biotechnol. 12:173–184; Lowman et al., (1991) Biochemistry 30(10):832–838; Lowman et al. and Wells J. (1993) J. Mol. Biol. 234:564–578; Dennis M. and Lazarus R. (1994) J Biol. Chem. 269(22):22137–22144.
The allergic response, when mediated by immunoglobulins of the E classification (IgE), is associated with disease states such as asthma and allergic rhinitis. The interaction between IgE and its high affinity receptor is a key step this allergic response. IgE immunoglobulins, specific for particular allergens, bind through their Fc region to specific high affinity receptors, FcεRI, on mast cells, basophils, and other cells (Ishizaka et al., (1970) Immunochemistry 7:687–702; Metzger et al., (1986) Ann. Rev. Immunol. 4:419–470; Kinet (1999) Ann. Rev. Immunol. 17:931–972). Antigen crosslinking of IgE bound FcεRI initiates an allergic cascade that results in mast cell degranulation and release of mediators of the allergic response such as histamine, leukotrienes and prostaglandins. Release of these mediators in turn leads to increased vascular permeability and the infiltration of platelets, eosinophils and neutrophils into surrounding tissue.
The FcεRI is present on cells as either a trimeric αγ2 or tetrameric αβ2 structure with the extracellular domains of the a chain conferring high affinity IgE binding. The crystal structure of the α chain has recently been solved (Garman S. C., et al. (1998) Cell 95:951–961). Mutagenesis studies have been performed to identify residues which contribute to IgE-Fc/FcεRI complex formation (Nissim et al., (1991) EMBO J. 10:101–107). Reports of the crystal structure of a complex of human FcεRI with bound IgE-Fc suggest two distinct binding sites for IgE-Fc in the IgE-Fc/FcεRI structure (Garman et al., (2000) Nature 406:259–266).
The binding of antibodies to the Fc portion of IgE can inhibit binding of IgE to receptor (Presta et al., (1993) J. Immunol. 151:2623–2632; Kolbinger et al., (1993) Protein Engineering 6:971–980) and can reduce free IgE levels in vivo (Saini et al., (1999) J. Immunol. 162:5624–5630). It has also been reported that certain peptides, designed to mimic a portion of the FcεRI receptor, can bind to the Fc portion of IgE and inhibit IgE binding to receptor (McDonnell et al., Nature Struct. Biol. 3:419425; McDonnell et al., (1997) Biochem. Soc. Trans. 25:387–392).
Presta et al. (1993) J. Immunol. 151:2623–2632 disclose a humanized anti-IgE antibody that prevents the binding of free IgE to FcεRI but does not bind to FcεRI-bound IgE. Clinical studies of allergic individuals using an anti-IgE monoclonal antibody have been reported (Jardieu and Fick (1999) Intl. Arch. Allergy Immunol. 118:112–115).
As a result, molecules which block the binding of IgE to FcεRI, the IgE high affinity receptor would also be expected to have efficacy in the treatment of IgE-mediated disorders.